Chromium removal and its association with rhamnolipid production in Pseudomonas spp. were investigated. Three Pseudomonas spp. isolates (P. aeruginosa 78, P. aeruginosa 99, and P. stutzeri T3) were investigated with regard to their exposure to 10mg/L for chromium removal. P. aeruginosa 99 removed 16% and 20% more chromium than P. stutzeri T3 and P. aeruginosa 78 respectively. The reduction of Cr(VI) to Cr(III) by all the three isolates is more or less similar. P. aeruginosa 99, which removed higher chromium, also produced higher rhamnolipid (165±5mg/mL). P. aeruginosa 78, which removed lower chromium, also produced lower rhamnolipid (126±3mg/mL). Rhamnolipid production by P. aeruginosa 78 and P. aeruginosa 99 was increased in its exposure to 10mg/L chromium. In the present study, results showed that rhamnolipid might play a role in chromium removal by three Pseudomonas spp. isolates.